Different LED Light Wavelengths and Photosynthetic Photon Flux Density Effect on Colletotrichum acutatum Growth

The study aimed to evaluate the effect of different photon flux density (PFD) and light-emitting diodes (LED) wavelengths on strawberry Colletotrichum acutatum growth characteristics. The C. acutatum growth characteristics under the blue 450 nm (B), green 530 nm (G), red 660 nm (R), far-red 735 nm (FR), and white 5700 K (W) LEDs at PFD 50, 100 and 200 μmol m−2 s−1 were evaluated. The effect on C. acutatum mycelial growth evaluated by daily measuring until five days after inoculation (DAI). The presence of conidia and size (width and length) evaluated after 5 DAI. The results showed that the highest inhibition of fungus growth was achieved after 1 DAI under B and G at 50 μmol m−2 s−1 PFD. Additionally, after 1–4 DAI under B at 200 μmol m−2 s−1 PFD. The lowest conidia width was under FR at 50 μmol m−2 s−1 PFD and length under FR at 100 μmol m−2 s−1 PFD. Various LED light wavelengths influenced differences in C. acutatum colonies color. In conclusion, different photosynthetic photon flux densities and wavelengths influence C. acutatum growth characteristics. The changes in C. acutatum morphological and phenotypical characteristics could be related to its ability to spread and infect plant tissues. This study’s findings could potentially help to manage C. acutatum by LEDs in controlled environment conditions.

Potential of Streptomyces sp. in preventing the in vitro growth of Colletotrichum acutatum, the causative agent of infection in Capsicum annum L.

Red chilli plant (Capsicum annum L.) is one of the most popular vegetable crops in Indonesian society. One of them the pathogens attacks is Colletotrichum acutatum, a fungus causing anthracnose on red chilli. This study aims to determine the existence of Streptomyces sp. bacteria in the rhizosphere of the red chilli plant; the ability of Streptomyces sp. in inhibiting C. acutatum; Minimum Inhibitory Concentration (MIC) of Streptomyces isolates extracts in inhibiting C. acutatum; The Streptomyces isolation was carried out by dilution method using selective meida, namely Yeast Malt Agar. The Dual Culture method was used as an inhibition test between Streptomyces sp. and C. acutatum in vitro. A well diffusion method was used to test the effectiveness of the Streptomyces sp. and MIC filtrate concentration in inhibiting C. acutatum. The data obtained in this study were analyzed with Analysis of Varian (ANOVA) then continued with Duncan Multiple Range Test with 5% significance. Five Streptomyces isolates were found, namely Streptomyces sp.1, Streptomyces sp.2, Streptomyces sp.3, Streptomyces sp.4, and Streptomyces sp.5 in the rhizosphere of healthy C. annum L. plants in Daup Village, Kintamani District, Bangli Regency. Streptomyces sp. isolates. can significantly inhibit the growth of the fungus C. acuatum with inhibitory power ranging from 50.30% to 83.76%, Streptomyces sp.5 isolate was able to provide the highest percentage of inhibition in C. acutatum of 83.76 ± 2.91% with MIC 7% (v/v) with a diameter of 6.40 mm.

First Report of Anthracnose Caused by Colletotrichum acutatum On Olive Fruits in Pakistan

Anthracnose symptoms on olive (Olea europaea) fruits cv. “Gamlick” were found in farmer orchards in Chakwal, Punjab, Pakistan (32° N and 72° E), with an average prevalence of 59%. Fruit symptoms start as thin, black, sunken lesions with a watery appearance that grow in diameter and coalesce into a large sunken soft zone. Lesions on mature fruit become noticeable in 5 to 6 days after infection, if temperatures are favorable (28°C). On the fruit lesion, orange conidial masses in dispersed or concentric circle arrangement can appear. Fragments (5 mm) were taken from the margins of fruit lesions and surface-sterilized with 70% ethanol (1 min) and 1% NaClO (2 min), cleaned with sterile purified water, blotted dry, and plated on potato dextrose agar (PDA) in Petri dishes. The petri plates were incubated at 27°C. A fungus was consistently isolated, and thirty-five isolates were characterized. Aerial mycelia from olive isolates Colonies were compact, initially white or cream white, then grey, and eventually dark grey, with conidium masses forming in the middle. Mycelium is branched, septate, and hyaline. Conidia are hyaline, aseptate, fusiform, or often cylindrical, with obtuse apices and tapering bases. Their mean size was 8.5µm in length and 3.0 µm in width. Based on morphological features, the fungus was tentatively identified as Colletotrichum acutatum (Agosteo G.E., 2010). The identification was confirmed by amplification and sequencing of a representative isolate's internal transcribed region (ITS), Beta- tubulin region (TUB2), Actin region (ACT), and Glyceraldehyde 3-phosphate dehydrogenase region (GAPDH) with the primers ITS1/ITS4 (Gardes & Bruns 1993), TUB4/TUB5 (Woudenberg et al. 2009), ACT1/ACT3 (Carbone & Kohn 1999) and GDF1/GDR1 (Guerber et al. 2003). BLAST analysis revealed 100% identity for ITS, GAPDH and ACT and 99% identity for TUB, between the sequences of the olive fruit isolate (GenBank Accessions MW647502, MZ436968, MZ714412 and MW810331, respectively) and sequences of C. acutatum reference isolates (GenBank Accessions GO613492, KF975660.1, MT274752.1 and MH547616 respectively). Phylogenetic analysis based on ITS, GAPDH and TUB regions of the olive fruit isolates and reference isolates of various Colletotrichum species using the MEGA X software program confirmed the isolate from olive was C. acutatum. The fungal isolate was deposited as a living culture in the Barani Agricultural Research Institute's fungal culture collection center (BACA.9381). Pathogenicity tests were conducted with this isolate by placing a 20 µl drop of a conidial suspension (3 × 107conidia ml−1) on five healthy olive cv. Gemlik fruits. As a control, five non-inoculated olive fruits were used. Fruits were placed at a temperature of 27°C with artificial light and a photoperiod of 12 hours. Anthracnose symptoms developed only on inoculated fruits after seven days of inoculation. The fungus was re-isolated from symptomatic fruits, and its identity was confirmed through morphological characteristics, thus verifying Koch's postulates. To the best of our knowledge, this is the first report of C. acutatum infecting olive fruits in Chakwal region of Pakistan.

Multitrophic Interactions Between Arbuscular Mycorrhizal Fungi, Foliar Endophytic Fungi and Aphids

Almost all living plants can be simultaneously colonised by arbuscular mycorrhizal fungi in the roots and endophytes in the shoots, while also being attacked by insect herbivores. However, to date, no study has ever examined the multitrophic interactions between these two different fungal groups and insects on any species of forb. Here, we examined the effects of two commercial species mixtures of arbuscular mycorrhizal fungi (AMF) and two foliar endophytes (Colletotrichum acutatum and Cladosporium oxysporum) on the growth of an invasive weed, Impatiens glandulifera, and the aphids that attack it. AMF reduced plant biomass, which was most evident when C. oxysporum was inoculated. Mycorrhizal fungi had few effects on aphids, and these depended on the identity of the endophytes present. Meanwhile, endophytes tended to increase aphid numbers, but this depended on the identity of the AMF inoculum. Throughout, there were differences in the responses of the plant to the two mycorrhizal mixtures, demonstrating clear AMF specificity in this plant. These specific effects were also strongly affected by the endophytes, with a greater number of interactions found between the AMF and endophytes than between the endophytes themselves. In particular, AMF reduced infection levels by the endophytes, while some endophyte inoculations reduced mycorrhizal colonisation. We suggest that both AMF and endophytes could play an important part in future biological control programmes of weeds, but further multitrophic experiments are required to unravel the complexity of interactions between spatially separated parts of the plant microbiome.

Growth promotion of raspberry and strawberry plants by bacterial inoculants

Study on potential mechanisms influencing the growth of raspberry and strawberry plants showed that the most active was Bacillus sp. strain AF75BC producing IAA and siderophores, and having the ability to release phosphorus. The latter feature was also present in the strains Sp115AD (B. subtilis) and SP116AC (Paenibacillus polymyxa). Two of the tested strains: SP116AC and JaFGU (Lysobacter sp.) showed the ability to fix atmospheric nitrogen, while the AF75AB2 (Bacillus sp.) produced siderophores and IAA. All strains showed an antagonism toward the most important pathogens of strawberry and raspberry, i.e. Verticillium dahliae, Botrytis cinerea, Phytophthora cactorum and Colletotrichum acutatum, limiting their growth to a different extent on the PDA medium. Inoculation of raspberry roots with the tested bacteria resulted in an increase of some growth parameters of their above-ground part in cv. Poemat. In the case of cv. Polana, a significant increase was found only in the chlorophyll content in the leaves. All the inoculants caused an increase in dry mass of roots in cv. Polana, and in cv. Poemat similar effect was observed after applying Inoculants 1 and 3. The treatments of strawberry roots with any of the inoculants resulted in a significant increase in the total leaf surface area in cv. Rumba, but they had no effect on the chlorophyll content in the leaves of either cultivar. All the inoculants significantly increased the total length of roots and their total surface area in cv. Rumba. This parameter also increased in cv. Elsanta, and the number of root tips also significantly increased in this cultivar. Our study showed that the tested inocula is a promising alternative as a bio-fertilizer for small fruit production in sustainable and organic agricultural systems.

Discovering the Next-Generation Plant Protection Products: A Proof-of-Concept via the Isolation and Bioactivity Assessment of the Olive Tree Endophyte Bacillus sp. PTA13 Lipopeptides

Endophytic microorganisms (EMs) have recently attracted interest for applications in plant protection, mainly due to their bioactive compound-producing capacity. Therefore, we underwent the task of isolating olive tree EMs and investigating their bioactivity against the devastating pathogen Colletotrichum acutatum. Several EMs were isolated; however, the Bacillus sp. PTA13 isolate exhibited the highest toxicity to the phytopathogen. Bacteria of the genus Bacillus exhibit superior bioactive metabolite-producing capacity, with the lipopeptides (LPs) of surfactin, iturin, and fengycin groups being the most studied. A total LP extract and several fractions were obtained, and their bioactivity was assessed against C. acutatum strains. LPs of the major surfactin, iturin, and fengycin groups and the minor gageotetrin and bacilotetrin groups were annotated. The results confirmed the bioactivity of the major LPs, with fengycins being the most fungitoxic. Interestingly, the minor LP fraction exhibited selective toxicity to the fungicide-resistant C. acutatum isolate, an observation that highlights the significance of our approach to comprehensively mine the total LP extract. This work represents a proof of concept of the exploitation of EMs in customized olive tree plant protection and aligns well with strategies that focus on the sustainability and safety of food production via the development of next-generation plant protection products.

Screening and Identification of Strains With High Lignocellulose-degrading Enzyme Production From Endophytic Fungi of Taxillus Chinensis

Taxillus chinensis is an extensively used medicinal herb in the traditional as well as modern systems of medicines. It is a perennial hemiparasitic plant, which is difficult to propagate artificially because of its low parasitic rate. Successful parasitism of parasitic plants is to fuse their tissues and connect their vasculature to the host vasculature building a physiological bridge, which can efficiently withdraw water, sugars and nutrients from their host plants. It is reported that endophytic fungi play an important role in cell wall degradation and fusion, which is the key forming process of the physiological bridge. Therefore, in this study, the endophytic fungi from T. chinensis of different hosts were isolated, and then the organisms that could degrade the main components of the cell walls were screened out using a medium consisting of guaihuol and cellulose degradation capacity. The results showed that five strains with high enzyme activities for lignocellulosic degradation were screened out from 72 endophytic fungi of T. chinensis. The laccase and cellulase activities of five strains reached their peaks at day 7, and the highest enzyme activities of these two enzymes were found in strain P6, which was 117.66 and 1.66 U/mL, respectively. Manganese peroxidase of strain 4 and lignin peroxidase of strain N6 also reached their peaks at day 7 and were the highest among the 5 strains, with enzyme activities of 11.61 and 6.64 U/mL, respectively. Strains 4, 15, 31, N6 and P6 were identified as Colletotrichum acutatum, Nigerrospora sphaerica, Exserohilum rofolium, Diaporthe phaseolorum and Pestalotiopsis arceuthobii, respectively, according to their morphological and molecular biology properties.

Effects of Exogenous Gibberellic Acid in Huanglongbing-affected Sweet Orange Trees under Florida Conditions – I. Flower Bud Emergence and Flower Formation

Under Florida conditions, sweet orange (Citrus sinensis) affected by Huanglongbing {HLB [Candidatus Liberibacter asiaticus (CLas)]} frequently exhibits irregular flowering patterns, including off-season flowering and prolonged bloom period. Such patterns can increase the opportunity for temporal and spatial proliferation of pathogens that infect flower petals, including the fungal causal agent for postbloom fruit drop (PFD) Colletotrichum acutatum J.H. Simmonds. For the development of strategies to manipulate flowering, the effects of floral inhibitor gibberellic acid (GA3) sprayed monthly at full- and half-strength rates (49 and 25 g·ha−1, or 33 and 17 mg·L−1, respectively) with different regimens, starting from September and ending in November, December, or January, on the pattern of spring bloom were evaluated in field-grown HLB-affected ‘Valencia’ sweet orange at two locations in subsequent February through April for two separate years in this study. To further examine whether GA3 effects on flowering patterns vary in different cultivars, early-maturing ‘Navel’ sweet orange trees receiving no GA3 or full-strength GA3 monthly in September through January were included. Overall, for ‘Valencia’ sweet orange, monthly applications of GA3 at 49 g·ha−1 from September to December not only minimized the incidence of scattered emergence of flower buds and open flowers before the major bloom but also shortened the duration of flowering, compared with the untreated control trees. In addition, exogenous GA3 led to decreased leaf flowering locus t (FT) expression starting in December, as well as reduced expression of its downstream flower genes in buds during later months. When applied monthly from September through January at 49 g·ha−1, similar influences of exogenous GA3 on repressing flower bud formation and compressing bloom period were observed in ‘Navel’ sweet orange. These results suggest that by effectively manipulating flowering in HLB-affected sweet orange trees under the Florida climate conditions, exogenous GA3 may be used to reduce early sporadic flowering and thereby shorten the window of C. acutatum infection that causes loss in fruit production.

First Report of Colletotrichum nymphaeae Causing Anthracnose on Almond in Hungary

Almond (Prunus dulcis [Mill.] D. A. Webb) is cultivated in commercial orchards in southwestern Hungary while numerous backyard orchards predominate in Buda Hills and central Hungary. In July 2019, anthracnose symptoms and necrotic twigs were observed across almond genotypes in a meadow orchard of Óbuda and in the genebank collection of the Hungarian University of Agriculture and Life Sciences. Fruits of some genotypes were damaged 100%, whereas others to a lesser degree or asymptomatic. Orange slightly sunken lesions on fruits produced gum. Near the diseased fruits the young shoots shriveled, the stalks became necrotized, on twigs necrosis developed. Isolates obtained from orange conidial masses from epicarp, necrotized tissues from twigs, and stalks were grown on PDA for 7 days at 25 ֯C in the dark. Upper surfaces of the colonies were white to pale gray, black solid mycelial structures were formed, the reverse side varied white to salmon. Acervuli were not formed, but conidia were produced from hyphae. Conidia were unicellular, hyaline, smooth-walled, cylindrical, predominant with both end rounded, or one end acute. Morphometric measurements of conidia showed mean length ± SD × width ± SD = 18.0 ± 2.2 × 4.7 ± 0.6 μm (n = 100). The isolates were morphologically identified as Colletotrichum acutatum sensu lato (Damm et al. 2012). Sexual morph was not observed. Three monosporic isolates were used for molecular identification. Partial nucleotide sequences were amplified from three loci, internal transcribed spacer (ITS), β-tubulin (TUB2) and calmodulin (CAL) after White et al. (1990); Glass and Donaldson (1995) and Weir et al. (2012), respectively. The ITS sequences (GenBank accessions MW425388 to MW425390) of the three isolates revealed that they belong to the C. acutatum species complex while BLAST results showed that TUB2 sequences (GenBank accessions MW428285 to MW428287) had 99.3% identity with C. nymphaeae strain CBS515.78, whereas the CAL sequences (GenBank accessions MW428288 to MW428290) had 100% with C. nymphaeae strain FREC138. The phylogenetic tree containing all the valid species of C. acutatum species complex confirmed that the isolates clustered to C. nymphaeae with high bootstrap support. The fungus was identified as Colletotrichum nymphaeae (Pass) Aa based on morphometric and molecular biological evidence. In vivo pathogenicity tests were conducted on ten healthy fruits, and ten twigs by inserting mycelial agar plugs (5 mm in diameter) onto wounded pericarp and phloem tissues, which were then wrapped in wet cotton and Parafilm®. The control treatments received sterile PDA discs. After 15 days, necrotic lesions 12 to 19 mm in diameter developed on fruit, 9 to 18 mm on twig. Control fruits, and twigs were asymptomatic. Koch’s postulates were fulfilled with the reisolation of the pathogen from symptomatic tissues. The ITS, ACT and CAL sequences of the reisolated Colletotrichum were determined and found identical to the original isolates. Anthracnose symptoms are known on almond fruits in several almond growing regions all over the word caused by Colletotrichum acutatum, C. godetiae, C. fioriniae, C. simmondsii, and C. gloeosporioides (Adaskaveg et al. 1997; López-Moral et al. 2000; de Silva et al. 2021; Shabi et al. 1983;). To our knowledge, this is the first report of Colletotrichum nymphaeae causing anthracnose of almond globally.